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J R Rogero

Publications and source records attributed to J R Rogero.

At least 19 recordsLinked to original sources
PubMed2006

Distribution of (125)I-labeled crotamine in mice tissues.

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with (125)I using chloramine T method, and separated by gel filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6)cpm/mouse) of (125)I native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain.

Animals↗
PubMed2001

Bothrops moojeni venom kills Leishmania spp. with hydrogen peroxide generated by its L-amino acid oxidase.

Leishmaniasis is an endemic tropical disease in South America, with few therapeutic approaches. Snake venoms are complex protein mixtures with biological actions that could be used as tools for drug development. Here we show that Bothrops moojeni crude venom presented a killing effect in vitro against Leishmania spp. promastigotes, but not with amastigotes, as determined by a viability assay using the mitochondrial oxidative function. Purification of active fractions from crude venom was performed by molecular exclusion and ion exchange chromatography. Anti-Leishmania and l-amino acid oxidase (L-AAO, EC.1.4.3.2.) activities co-eluted in the same fractions. The molecular weight of the active enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa by SDS--PAGE, with a 4.8 isoelectric point. Using substrate subtraction and catalase for scavenging, the action of L-AAO was demonstrated to be hydrogen-peroxide-dependent.

Amino Acid Oxidoreductases↗
PubMed2001

Gyroxin fails to modify in vitro release of labelled dopamine and acetylcholine from rat and mouse striatal tissue.

Gyroxin fails to modify in vitro release of labelled dopamine and acetylcholine from rat and mouse striatal tissue. Gyroxin is a thrombin-like peptide with amidasic, esterasic and fibrinogenolitic activities, found in the venom of snakes like Lachesis muta muta and Crotalus durissus terrificus. Intravenous injections of small doses of gyroxin induce a typical barrel rotation behaviour that has been thought to be a neurotoxic effect. The aim of this study was to determine whether gyroxin-induced barrel rotation behaviour involves changes in neurotransmitter release. Gyroxin was isolated from crude venoms by gel filtration and affinity chromatography. Its properties were determined by assaying esterasic, amidasic and fibrinogenolitic enzymatic activities and tested for barrel rotation behaviour. Neurotransmitter release tests employed rat and mouse superfused brain striatal chopped tissue preloaded with [(3)H]-dopamine, [(3)H]-acetylcholine or in a double labelling procedure. They were stimulated by 20mM K(+) in control conditions or in the presence of several concentrations of toxins. Crotoxin and crotamine were used as positive controls. Gyroxins failed at modifying both basal and stimulated neurotransmitter releases, suggesting a lack of direct neurotoxic effect. We therefore suggest that gyroxin may not be a neurotoxin but rather, induces this behavioural syndrome by other means possibly related to haemodynamic disturbance. The possible role of vasopressin is discussed.

Acetylcholine↗
PubMed2001

Effects of (60)Co gamma radiation on crotamine.

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Animals↗
PubMed2000

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission.

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.

Animals↗
PubMed2000

Gabaergic-benzodiazepine system is involved in the crotoxin-induced anxiogenic effect.

The behavioral effects of crotoxin (CTX), the major component of Crotalus durissus terrificus venom, were studied in rats submitted to the open field, holeboard, and social interaction tests. CTX (100, 250, and 500 microg/kg, i.p.) was administered 2 h before the tests. In the open field, CTX reduced ambulation (250 microg/kg) and rearing (250 and 500 microg/kg) and increased grooming (100 and 250 microg/kg) and freezing (250 microg/kg). In the holeboard and social interaction, all the CTX doses evaluated decreased, respectively, head dip and head dipping, and social interaction time. The CTX-induced behavioral alterations could be attributed to its neuromuscular transmission blockade, but this possibility was ruled out because CTX (250 and 500 microg/kg, i.p., 2 h before the rotarod test) was unable to modify the rotarod performance of rats. The involvement of the benzodiazepine receptor in the CTX-induced behavioral alterations was investigated through the pretreatment (30 min before the tests, i.p.) of the animals with diazepam (1.2 mg/kg), or flumazenil (4 and 10 mg/kg). Both diazepam and flumazenil antagonized the CTX-induced behavioral alterations in the open field, holeboard, and social interaction tests. This study demonstrated that: (1) CTX is an anxiogenic compound; and (2) the gabaergic-benzodiazepine system may play a role in the CTX-induced anxiogenic effect.

Animals↗
PubMed1999

Toxicity and immunogenicity of Crotalus durissus terrificus venom treated with different doses of gamma rays.

Crotalus durissus terrificus venom (CDT venom) was irradiated with four different doses of gamma rays (2, 3, 5 and 10 kGy) from a 60Co source and their structural, toxic and immunogenic properties were analysed. Venom irradiated with 2 and 3 kGy were, respectively, 2.7 and 13.5 times less toxic than the native one, whereas the 5 or 10 kGy irradiated venom were at least 100 times less toxic than nonirradiated venom. Irradiated venom with all doses were immunogenic and the antibodies elicited by them were able to recognise the native venom in ELISA. However the toxoid produced with 2 kGy irradiation dose had its immunogenicity improved. Antisera raised against this toxoid had a higher neutralising capacity than those produced against the native venom. Irradiation of venom with 2 kGy dose was the most effective to inactivate the CDT venom toxicity and improve its immunogenicity.

Animals↗
PubMed1998

Differential biodistribution of native and 2 kGy 60Co irradiated crotoxin in tissues of CBA/J mice.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Animals↗
PubMed1998

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation.

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 microg/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum.

Animals↗
PubMed1998

A single-step purification of bothropstoxin-1.

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Animals↗
PubMed1997

Effect of gamma irradiation on the behavioral properties of crotoxin.

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g) were used. Crotoxin (100, 250, and 500 micrograms/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 micrograms/kg for the hole-board test, and at the doses of 1000 and 2500 micrograms/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P < 0.05). Crotoxin decreased the social interaction time(s) at the doses of 100, 250 and 500 micrograms/kg (means +/- SEM) from 51.6 +/- 4.4 to 32.6 +/- 3.7, 28.0 +/- 3.6 and 31.6 +/- 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.

Animals↗
PubMed1996

Influence of ionizing radiation on crotoxin: biochemical and immunological aspects.

Irradiation of crotoxin and its subunits with 2000 Gy of gamma-rays from 60Co source leads to aggregation and generation of lower mol. wt breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformational epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-react and neutralize crotoxin. Compared with native crotoxin, aggregates appear less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin.

Animals↗
PubMed1996

Crotoxin-induced behavioral effects in rats.

Crotoxin is the major component of Crotalus durissus terrificus venom. In view of the presence of high-affinity specific binding sites for crotoxin in the brain, the objective of this work was to investigate whether crotoxin induces behavioral effects in the open-field and hole-board tests. Adult male Wistar rats (180-220 g) treated with crotoxin, 100, 250 and 500 micrograms/kg, ip, administered 2 h before the test, presented statistically significant behavioral alterations (ANOVA for one-way classification complemented with Dunnet test, P < 0.05). In the open-field test, 250 and 500 micrograms/kg of crotoxin increased freezing (from 3.22 sec to 10.75 sec and 11.2 sec) and grooming (from 13.44 sec to 22.75 sec and 21.22 sec) and decreased ambulation (from 64.8 to 39.38 and 45.8). The dose of 500 micrograms/kg also decreased rearing (from 24.9 to 17.5). In the hole-board test, 500 micrograms/kg of crotoxin decreased head-dip count (from 6.33 to 4.00). All the crotoxin-induced behavioral effects were antagonized by an anxiolytic dose of diazepam (1.5 mg/kg, ip. 30 min before the tests). These results show that crotoxin reduced open-field activity and exploratory behavior as well. We suggest that these effects express an increased emotional state induced by this toxin.

Analysis of Variance↗
PubMed1992

Detoxification of the crotoxin complex by gamma radiation.

1. Crotoxin, the major neurotoxin of the South American rattlesnake venom (2 mg protein/ml in 0.85% NaCl) was irradiated with a Co-60 gamma source at a dose rate of 1100 Gy/h and at doses of 250, 500, 1000, 1500 and 2000 Gy. 2. Irradiated crotoxin was analyzed for free SH-groups, protein concentration, electrophoretic profile (SDS-PAGE), 50% lethal dose (LD50) in mice, and antigenicity against crotalic antiserum by diffusion immunoassay. 3. Irradiation led to the formation of protein aggregates and solubility was reduced at doses of 1000 Gy or higher. 4. The LD50 increased about 2-fold for 1000 Gy and 3.5-fold for 1500 Gy. However, the antigenic response was not changed as judged by the capacity of irradiated protein receiving up to 1000 Gy to react with anti-Crotalus durissus terrificus venom horse serum. 5. The dose of 1000 Gy cleaved 0.95 disulfide bridges/mol and 1500 Gy cleaved 1.42 bridges/mol, indicating the importance of disulfide bond integrity for toxicity.

Animals↗
PubMed1992

Immunochemical detection of purified crotoxin from Crotalus durissus terrificus venom in the motor end plate of striated muscle in CBA/J mice.

Electrophysiological studies of crotoxin, a potent neurotoxic fraction from Crotalus durissus terrificus venom, have shown a pre- and post-synaptic action. In order to determine the specific site of this activity, we performed an immunohistochemical analysis on striated muscle from CBA/J mice, injected with crotoxin (5 LD50), iv, using a single-step immunoperoxidase assay with peroxidase-conjugated antibodies to whole venom. Control muscle and muscle obtained from treated animals 15 min after injection showed no staining. However, 30 min after injection, the neuromuscular motor end plate of mice who received crotoxin was clearly stained, including thin terminations, without any morphological alteration. Sixty min after administration, the motor end plate was no longer intact, but only roughly formed stained areas without clearly identifiable structures were present. These data show specific crotoxin binding to neural end plates in striated muscle, with a subsequent toxin-induced degeneration of this structure.

Animals↗
PubMed1991

Pathophysiology of Haemonchus placei infection in calves.

This study was conducted to investigate the pathophysiology of Haemonchus placei infection in Friesian calves. Seven calves were divided into two groups, three uninfected calves (control group) and four infected animals. The latter group were infected orally with 500 H. placei larvae kg-1 body weight. Five weeks after infection they were all housed in metabolic crates and injected with 125I-bovine albumin. 51Cr-red cells and 59Fe-transferrin, to study albumin metabolism, erythrokinetics and ferrokinetics. The results showed that there was a significant reduction in the mean haematocrit values and reduced weight gains in the infected calves compared with the controls. There was also a change in the distribution of albumin from the extravascular to the intravascular pool and a significant increase in the plasma and blood volumes of infected calves although the blood and albumin loss via the gastrointestinal tract recorded in this study was similar in both groups.

Age Factors↗